Evidence is presented in support of the hypothesis that in the hydrolysis of DPN by the DPN nucleosidase of rabbit erythrocyte stroma, the substrate is attached to the enzyme at more than one site. Cleavage of DPN at the nicotinamide–ribose bond is inhibited by nicotinamide, ADP, and adenine. Considering the nature of the inhibition and the extent of the inhibition, it is suggested that DPN attaches to the enzyme surface at the quaternary nitrogen of the nicotinamide and at the pyrophosphate group of the DPN molecule. Additional support for this supposition accrues from the observation that NMN, a cleavage product of DPN, can serve as a substrate for DPN-ase but the rate of hydrolysis is much slower than that with DPN.